Mammalian cells are usually cultured in glass or plastic vessels, either in suspension or as an attached layer, completely surrounded by culture media. When nutrients are depleted or metabolic waste products accumulate, spent growth media is exchanged for fresh to sustain the culture (feeding, see Dexter, T. M., "Cell Interactions in Vitro", Clinics in Haematology, 8, 453-568 [1979]). Consequently, cells experience periodic shifts in nutrient concentrations ranging from feast (fresh media) to famine (spent media) during the course of a culture period which can last several days to a few weeks. Likewise, pericellular pH varies with a refeeding schedule because fresh media is basic relative to spent due to accumulation of acidic waste products during culture. Furthermore, soluble macromolecular cell products such as attachment factors, antibodies, and hormones (termed extracellular material or ECM; Wilde, C. E., "Bull. Groupe Franc. Argiles", 15, 183 [1961]) are removed with each refeeding, potentially altering cellular biosynthetic processes.
Although these periodic perturbations of the culture environment are not detrimental for relatively short term culture, long-term culture of cells in vitro requires a stable environment and retention of ECM (see Rose, G. G., "Cytopathophysiology of Tissue Cultures Growing under Cellophane Membranes", International Review of Experimental Pathology, Richter, G. W., Eptsein, M. A. eds., Vol. 5, 111-174, Academic Press [1966]).
In order to diminish these environmental fluctuations, cells and tissue have been grown under contiguous or perforated sheets of cellulosic film so that at least part of the cellular milieu persists after media exchanges (for a review of the subject see Rose, G. G., ibid). Moreover, putative low molecular weight inhibitors and metabolic waste products can dialyze out of cell growth space. Although this method is an advancement in long-term culturing of attached cells or tissue, refeeding is still required and perturbations in cellular environment not totally avoided. Furthermore, the method is not applicable to suspension cells such as hybridoma lines because loose cells are not retained under unbounded edges of the cellulose membrane overlayer.
Simultaneous cell growth with continuous dialysis was developed by Marbrook (Marbrook, J., "Primary Immune Response in Cultures of Spleen Cells". The Lancet, 2, 1279-1281 [1967]). In this case two concentric chambers are separated by a dialysis membrane. The inner-growth chamber is partially submerged in the outer-chamber dialysis solution of larger volume. Cells are grown directly on the dialysis membrane within the inner-chamber and are continuously bathed in nutrients passing through the membrane from the outer-chamber reservoir. Disadvantages of this system include clogging of the dialysis membrane with cell mass, cellular debris, and ECM.
U.S. Pat. No. 4,296,205, issued Oct. 20, 1981 to Verma, D. S. describes the improvement over the Marbrook system of introducing a tissue-culture shelf held above the dialysis membrane to help prevent clogging and problems associated therewith. Fluid communication with the dialysis membrane and, ultimately, with a media reservoir is through perforations in the culture shelf. Although attached cells or tissue remain on the culture shelf, unattached cells and cellular debris, can become suspended and pass through tissue-culture-shelf perforations and clog or otherwise reduce critical membrane performance. Consequently, culture of suspension cells is not possible. Furthermore, during long-term culture, soluble ECM can crystallize into variously sized particles (termed biologic crystals and particles or BCP by Rose, G. G., ibid) onto or into the membrane, further compromising performance. Other difficulties with the Verma method include inability to adequately view cells during culture with high-power cytological phase-contrast and interference-contrast optics. Finally, equilibration of the culture media with incubator gasses, which is required to provide oxygen and carbon dioxide to the culture, occurs only through loosened flask caps. Proper ventilation is critical to maintenance of media pH because media mixes typically utilize a carbon dioxide/bicarbonate buffer system. Since the media reservoir volume is high, gas exchange through loosened caps is inadequate to rapidly equilibrate the entire media volume with the incubator atmosphere.
The art has sought a device and method that allows long-term, in vitro culture of both attached and suspension cells, simultaneously providing adequate gas ventilation, continuous supply of pH-equilibrated nutrient fluids, and the capability to microscopically view cells at any time during the course of culture. As shown herein, the disclosed invention meets these needs.